The Project (Transcription-Coupled and Replication-Associated Excision Repair) focuses on mechanisms coupling DNA excision repair machinery with transcription and replication. Both Nucleotide Excision Repair (NER) and Base Excision Repair (BER) are highly coordinated by interactions between proteins in the pathway. Moreover, they are preferentially targeted by specialized transcriptioncoupled repair (TCR) machinery to lesions that affect transcription elongation or by replication-associated repair (RAR) to lesions near the replication fork or in recently replicated DNA. We hypothesize that these interactions and their effects on function are regulated through unstructured flexible regions that undergo disorder-to-order transformations upon complex formation and/or post-translational modifications. We will test this overall hypothesis and specific hypotheses in five Aims by collaborative studies to characterize, validate, and map interactions, identify damage-induced modifications, observe effects of complexes on DNA structure by scanning force microscopy (SFM), and visualize subunits and complexes by electron microscopy (EM), small angle X-ray scattering (SAXS), and protein crystallography (PX). Aim 1 will structurally characterize early steps of TCR: recognition by XPG and CSB of RNA Polymerase II (RNAPII) stalled at a lesion, and remodeling of RNAPII by TFIIH to allow access to the lesion. SFM and EM studies will test the hypothesis that these occur by ordered conformational changes. Aim 2 will structurally characterize CSB and reinvestigate its causal role in CS by determining whether mutant CSB interferes with responses to oxidative DNA damage through non-productive interactions with other proteins in the pathway. Aim 3 will investigate the identified interactions that couple BER and NER to transcription through (a) SAXS and PX studies of XPG protein and its domains and complexes, (b) analysis of interactions of NEIL2 with RNAPII, XPG and CSB, and (c) characterization of the effect of post-translational modifications on XPG and NEIL2 interactions. Aim 4 will characterize the structural basis for BER pathway coordination by interactions of NEIL1 and NEIL2 glycosylases with downstream BER proteins and test the hypothesis that BER pathway progression results in progressive DNA bending. Aim 5 will investigate molecular mechanisms of RAR by determining the structure of the checkpoint sliding clamp -- the 9-1-1 complex - and by characterizing interactions of the MYH and NEIL1 glycosylases with PCNA and 9-1-1. The anticipated outcome is a molecular understanding of cancer predispositions and developmental disorders that arise from defects in the coordination of excision repair with transcription and replication. Collaborations of Project 2 within SBDR and with the UCSF Comprehensive Cancer Center will relate results of these studies to genome integrity and cancer etiology as well as to development of promising molecular targets for cancer drug discovery.